The present invention relates to a method for the fluorescence analysis of multiply stained particles, particularly biological cells, in continuous flowthrough in which the particles are suspended in a carrier fluid, are then enclosed in an envelope, or sheath, stream and are conducted in laminar flow through an intensive laser light zone, where the fluorescent light pulses emanating from the particles are detected and processed in real time by an electronic evaluation system.
Methods of this type are described, for example, in the published article by H. Crissman, P. Mullaney and J. A. Steinkamp, entitled "Methods and Applications for Flow Systems for Analysis and Sorting of Mammalian Cells", in the publication Methods in Cell Biology, Volume 9, pp. 179-246 (1975), published by D. M. Prescott. The basic structure of the measuring arrangement is illustrated in a brochure entitled "Laser Review" of the firm Spectra-Physics, Mountain View, California, under the heading "New Laser System Used in Biomedical Studies".
If a plurality of components of a cell are to be analyzed simultaneously, particularly with respect to its chromosome content (DNA) and its protein content, the sample must be stained with different fluorescent dyes which each react specifically with one of the components, i.e. the DNA or the protein. It is known to use dyes for this purpose which have different fluorescence emission spectra at the same excitation frequency. However, this entails a certain limitation in the selection of suitable dyes, which may have a negative effect mainly on the sample preparation time involved and on the detection sensitivity. In addition, this technique requires the use of semitransparent mirrors and separating filters which also bring about intensity losses for the pulses received by the detectors.